Multiplex polymerase chain reaction (Multiplex PCR) is a modification of PCR in order to rapidly detect deletions or duplications in a large gene. In this process, genomic nucleic acid samples are amplified using multiple primers and a temperature-mediated polymerase in a thermal cycler.
Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon size, that is, their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
Commercial multiplexing kits for PCR are available and used by many forensic laboratories to amplify degraded DNA samples. Commercial kits have a number of advantages over in-house multiplexing methods. Quality control measures are undertaken by the manufacturer of the kit and ensure that reactions are uniform across all kits. This avoids the preparation of PCR master mixes which require pipetting and use of multiple assay tubes, increasing the risk of operator error and contamination. This increased reliability allows profiles obtained from commercial kits to be admitted into court which is pivotal in large criminal trials. The use of specific kits over a number of laboratories also allows for profile results to be compared as long as the same STR markers have been used in each kit.
Some of the applications of multiplex PCR include: Pathogen Identification; High Throughput SNP Genotyping; Mutation Analysis; Gene Deletion Analysis; Template Quantification; Linkage Analysis; RNA Detection; Forensic Studies. The current systems available are very big and heavy machines. The machines are not able to do both real time PCR and multiplexing. The size and fragility of current systems means they are lab based and not able to be used in field conditions.
Current devices for multiplexing require several hundreds of watts to operate and around 75% of this energy goes into the heating and cooling of samples. Such high energy requirements limit the current machines to laboratory based settings where access to a continuous energy supply is available.
Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR/qrt-PCR) or kinetic polymerase chain reaction (KPCR), is a technique based on PCR, which is used to amplify and simultaneously quantify a targeted nucleic acid molecule. Real Time-PCR enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes.
The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time. For standard PCR the product of the reaction is detected at its end. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent intercalating dyes that disrupt any double-stranded DNA, and (2) sequence-specific nucleic acid probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary nucleic acid target.
The object of the invention is to alleviate some of the problems of the current devices and methods.